The enzymatic hydrolysis process, especially the saccharification of lignocellulosic biomasses, takes place in general in buffered media or under controlled pH. Thus, it is common to use buffering solutions, such as for example, sodium citrate and sodium acetate, among others, or pH control solutions such as acids and bases (e.g. sodium hydroxide, hydrochloric acid, phosphoric acid and etc) so as to maintain the pH in an ideal value in which the saccharification process of these biomasses is favored. In general, the optimum operation pH of the system matches to the optimum operation pH of the enzymes responsible for the bioconversion process of the biomass into fermentable sugars. Cellulase, the most used enzymatic complex in the saccharification process of lignocellulosic biomasses, expresses its major activity in the pH range that ranges from 4.5 to 5.5. This pH range, besides favoring the catalytic process, also helps in maintaining the stability of the enzymatic complex for longer time periods, which extends the shelf life of the biocatalyst impacting beneficially and in a crucial manner the final cost of the process.
Conventional buffering solutions such as sodium citrate and sodium acetate are very expensive and can also make the productive process impossible to be done in certain situations. Acids and/or bases used for the pH control during the process despite presenting a significantly lower cost in relation to before mentioned solutions still represent a significant portion of the final cost of the process when compared to the process using stillage as the buffering agent. Thus, it the use of effective and cheap buffering agents becomes extremely desirable in the aforementioned process.
The use of stillage in the saccharification process of lignocellulosic biomasses presents many advantages when compared to conventional processes, but certainly an advantage of great importance is the significant savings on water consumption in the step of enzymatic hydrolysis of biomasses (pretreated or not), once the stillage can also substitute all necessary water in this specific process.
The presence of stillage in the enzymatic hydrolysis process of lignocellulosic biomasses allows the formation of chemical compounds which have the ability to promote the buffering effect of the reaction medium, that is, to maintain the pH inside a specific desirable range in order to provide a favorable environment to the suitable functioning of the enzymes making part of the enzymatic complex, which need well defined conditions to perform the breaking of bonds of their specific substrates, in order to maximize the generation of fermentable sugars from the lignocellulosic biomass used.
In the buffering process of the reaction medium, salts present in the stillage such as, for example, sodium, match to acids generated during the pretreatment process of the biomass, such as, for example, acetic acid, forming chemical compounds, such as for example, sodium acetate, which directly acts on the maintenance of the medium pH in certain ranges. Hence, the buffering effect arises from the chemical reaction which takes place between compounds naturally present in the stillage and in the pretreated biomass, dispensing thus the advance preparation of the buffering solution.
The present invention has as main advantages:                greatly effective in the buffering of the reaction medium where the saccharification of lignocellulosic biomasses takes place, that is, the use of the stillage allows the pH of the suspension to be maintained in the optimum range of the enzymatic complex operation used (4.5-5.5), once biological catalysts express their major activity in a very strict range of the pH. Also, the maintenance of the pH in the optimum range of operation of the enzyme allows enzymes to maintain their stability for longer time periods, which extends the shelf life of the biocatalyst impacting in a beneficial and crucial way the final cost of the process;        cheaper than conventional buffering solutions such as, for example, buffer solutions sodium citrate and sodium acetate, as the current is produced inside the industrial unit in great amounts since the equivalent for each liter of produced ethanol it is generated around 10 to 12 liters of stillage;        rich in nitrogen, which is a macro nutrient of great importance in the fermentation process of sugars into ethanol, given that a typical industrial stillage can present until 1 g/L of total nitrogen in its composition; thus, besides cheapening the global cost of the process, the stillage still favors the specific step of fermenting hydrolyzed broths derived from the saccharification of sugar cane bagasse;        suitable for use in the saccharification process of lignocellulosic biomasses also under an environmental viewpoint following the concept of closed cycle process, since this “residue” or “byproduct” is commonly used in the sugarcane fertigation process, being a very important source of soil and ground water pollution mainly due to its increasing high biochemical oxygen demand (BOD) in accordance with organic matter content present in its composition;        allow water saving in the saccharification process of sugarcane bagasse substituting this one for the stillage.        
In the patent scope, some relevant documents have been identified, which will be discussed hereinafter.
The document U.S. Pat. No. 7,807,419 discloses a process for saccharification of pretreated biomass in order to obtain high concentrations of fermentable sugars. Specifically, it is addressed to a process using a batch wise feeding system having a reduction in the size of particles. The present invention differs from this document by comprising the use of the stillage as the buffering agent of the saccharification reaction of biomasses, as well as the additive to fermentation due to the nitrogen content present in its composition.
The document US 2010/0086981 presents compositions and methods to improve the saccharification of biomasses with enzymes and modified microorganisms. Said document mentions the use of sodium citrate during the reaction. The present invention differs from this document by dispensing the use of sodium citrate, substituting it by the available stillage in large volumes in the sugar cane mills.
The document US 2010/0159515 describes a method of pretreatment of lignocellulosic biomasses mentioning the use of a buffer from citrates. The present invention differs from this document by using compounds present in the available stillage in the alcohol and sugar mills or independent distilleries to promote the buffering effect to the reaction medium.
The document WO 2008/040358 describes the use of a fermentation byproduct, the stillage, which is rich in potassium as the fertilizing agent. This document also says that the stillage rich in potassium is poor in amino acids and proteins, since the same have already been removed in previous steps. The present invention differs from this document by previewing the use of the stillage resulting from the industrial alcohol fermentation rich in nitrogen (to 1 g/L) not as the fertilizing agent, but as the buffering agent of the saccharification process of lignocellulosic biomasses; in addition, this stillage also shows itself suitable to the step of alcoholic fermentation, since it represents an additional source of nitrogen, essential macro nutrient to said fermentation process.
The document EP 048061 describes a process for recycling the stillage comprising the steps of concentration and burning of the stillage. However, nothing is said about its use in hydrolytic (acids or enzymatic) and/or fermentation processes; neither is about its characteristics as buffering agent or source of nitrogen.
The document WO 2008/116278 describes the stillage recycling as additive in animal feed, the stillage passing through a process of distillation and concentration, followed by dehydration.
The document EP 769915 describes the use of organic acids derived from the stillage; such compounds prove especially useful in microbial crops.
The present invention differs from this and from the other documents by using the stillage (or any residue/byproduct derived from the fermented wine distillation process) in any state of treatment (raw, filtered, concentrated, etc) as buffering agent of saccharification processes of lignocellulosic biomasses and fermentation additive (additional source of nitrogen).
What is known from the literature is that no documents were found previewing or suggesting the teachings of the present invention, in such a way that the solution proposed here is new and has inventive activity in relation to the state of the art.